As a suggestion:
1. Perform your lysozyme treatment in buffer with 20% sucrose. 2. Then pellet your still intact cells and remove supernatant (cells pellet is white and cell lack walls - sphaeroplasts). Periplasmic proteases, metallophores, etc are all washed away. 3. Then resuspend in your lysis buffer and sonicate - cells are very fragile and burst easily. For more details, see supp methods of this paper: Nat Methods. 2009 Jul;6(7):477-8. Enabling IMAC purification of low abundance recombinant proteins from E. coli lysates. Magnusdottir A, Johansson I, Dahlgren LG, Nordlund P, Berglund H. PMID:19564847 [PubMed - indexed for MEDLINE] Good luck, Darren On 21 June 2012 14:44, J. Valencia S. <valen...@gene.nagoya-u.ac.jp> wrote: > Greetings, everyone. We need to ask your advice on an issue with one of > our proteins expressed in E. coli Rosetta cells. This yeast-derived > protein has a very low yield compared to others we work with, and we think > it is because the cells are hard to lyse: even after 3 cycles in a cell > cracker the solution barely changes colour. > > We have no problems lysing Rosetta cells expressing other yeast-derived > soluble proteins, and we usually obtain enough for our crystallisation > screens. For the aforementioned protein we have already tried using STAR > cells, varying the contents of the lysis buffer, sonicating, or adding > FeSO4 to the solution (we think the protein binds Fe or Mn because it is > yellow), but to no avail. > > Searching the ccp4bb archive and other resources did not help, so we would > like to ask 2 questions to the community in order to focus our efforts > better: > 1. How can a recombinant protein make a cell harder to lyse? > 2. Do you have any suggestions to avoid this effect? > > We appreciate any input, and will be sure to post a summary for future > reference once this issue is solved. > > Sincerely, > > > -- > J. Valencia S. > PhD student > CGR-NU > -- ********************************************************************** Dr. Darren Hart, Team Leader High Throughput Protein Lab Grenoble Outstation European Molecular Biology Laboratory (EMBL) ********************************************************************** www.embl.fr/research/unit/hart/index.html For funded access to ESPRIT construct screening via EU FP7 PCUBE: http://tinyurl.com/ydnrwg4 Email: h...@embl.fr Tel: +33 4 76 20 77 68 Fax: +33 4 76 20 71 99 Skype: hartdarren Postal address: EMBL, 6 rue Jules Horowitz, BP181, 38042 Grenoble, Cedex 9, France **********************************************************************
